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A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3.

Foster TL, Tedbury PR, Pearson AR, Harris M - Biochim. Biophys. Acta (2009)

Bottom Line: The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage.We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage.Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

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Related in: MedlinePlus

Comparative analyses of recombinant WT NS2-3 precursor proteins derived from J4 or JFH-1 isolates. (A) The cleavage activity of the J4 and JFH-1 precursors was probed by western blotting using anti-NS3 polyclonal sera and anti-FLAG monoclonal antibody. Genotype 2a JFH-1 NS2-3 lacks the truncation product of genotype 1b J4 NS2-3 detected by anti-NS3 antibody. The black triangle indicates the NS3 cleavage product while the red triangle indicates the truncation product observed in J4 NS2-3. (B) Sequence alignment from Clustal W2 multiple alignment of genotype 1b strain J4 NS2-3 (GenBank accession no. AF054250) and genotype 2a strain JFH-1 NS2-3 (GenBank accession no. AB047639). The tryptophan fluorophores are highlighted in yellow and the cleavage site in cyan and active site residues in magenta. (C) The pH of the cleavage buffer was varied by 0.5 pH units within the range from pH 6.0 to pH 9.0 to assess the dependence of cleavage activity on pH for the two proteins. (D) The temperature dependence of cleavage activity between J4 and JFH-1 NS2-3 was compared over a 16-h period. Optimal activity at 24 °C for both proteins and inactivity of JFH-1 at 4 °C compared to J4 as observed. (E) The inhibition of auto-processing of NS2-3 by the chelation of metal ions was assessed by the addition of EDTA, EGTA and 1,10-phenanthroline (1,10-phen) to the cleavage reactions at 1 mM concentrations. (F) Wild-type NS2-3 precursor and the two active site mutants H956A and C997A were expressed and purified by nickel affinity chromatography and analysed by 15% SDS–PAGE followed by Coomassie staining. For auto-processing, purified wild-type NS2-3 precursor or the two mutants H956A and C997A were supplemented with 100 mM DTT, diluted 1:100 into cleavage buffer and incubated at 25 °C or 4 °C. For all data in panels C–F apart from the Coomassie stained gel in panel F, the products were separated by SDS–PAGE and probed by western blotting with anti-FLAG monoclonal antibody. Black triangles indicate the NS2-3 precursor and the NS3 protease domain cleavage product.
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fig2: Comparative analyses of recombinant WT NS2-3 precursor proteins derived from J4 or JFH-1 isolates. (A) The cleavage activity of the J4 and JFH-1 precursors was probed by western blotting using anti-NS3 polyclonal sera and anti-FLAG monoclonal antibody. Genotype 2a JFH-1 NS2-3 lacks the truncation product of genotype 1b J4 NS2-3 detected by anti-NS3 antibody. The black triangle indicates the NS3 cleavage product while the red triangle indicates the truncation product observed in J4 NS2-3. (B) Sequence alignment from Clustal W2 multiple alignment of genotype 1b strain J4 NS2-3 (GenBank accession no. AF054250) and genotype 2a strain JFH-1 NS2-3 (GenBank accession no. AB047639). The tryptophan fluorophores are highlighted in yellow and the cleavage site in cyan and active site residues in magenta. (C) The pH of the cleavage buffer was varied by 0.5 pH units within the range from pH 6.0 to pH 9.0 to assess the dependence of cleavage activity on pH for the two proteins. (D) The temperature dependence of cleavage activity between J4 and JFH-1 NS2-3 was compared over a 16-h period. Optimal activity at 24 °C for both proteins and inactivity of JFH-1 at 4 °C compared to J4 as observed. (E) The inhibition of auto-processing of NS2-3 by the chelation of metal ions was assessed by the addition of EDTA, EGTA and 1,10-phenanthroline (1,10-phen) to the cleavage reactions at 1 mM concentrations. (F) Wild-type NS2-3 precursor and the two active site mutants H956A and C997A were expressed and purified by nickel affinity chromatography and analysed by 15% SDS–PAGE followed by Coomassie staining. For auto-processing, purified wild-type NS2-3 precursor or the two mutants H956A and C997A were supplemented with 100 mM DTT, diluted 1:100 into cleavage buffer and incubated at 25 °C or 4 °C. For all data in panels C–F apart from the Coomassie stained gel in panel F, the products were separated by SDS–PAGE and probed by western blotting with anti-FLAG monoclonal antibody. Black triangles indicate the NS2-3 precursor and the NS3 protease domain cleavage product.

Mentions: Previous biochemical studies of the NS2-3 protease have been performed with protein of the genotype 1b J4 isolate. One complication of the analysis of the activity and folding of J4 NS2-3 is the presence of a 30-kDa C-terminal truncation product of the full-length protease (Fig. 2A) [13], which is detectable with NS3 antisera but not detectable with the FLAG antisera, as the truncation lacks the FLAG-tag located at the C-terminus of the full-length protein. This abundant truncation product is non-functional but would contribute significantly to the data collected in analyses of the global folding of the sample by tryptophan fluorescence. This truncation product, however, is not detectable in preparations of JFH-1 NS2-3, making the NS2-3 protease from genotype 2a more suitable for the subsequent assessment of refolding of the functional, full-length precursor state.


A comparative analysis of the fluorescence properties of the wild-type and active site mutants of the hepatitis C virus autoprotease NS2-3.

Foster TL, Tedbury PR, Pearson AR, Harris M - Biochim. Biophys. Acta (2009)

Comparative analyses of recombinant WT NS2-3 precursor proteins derived from J4 or JFH-1 isolates. (A) The cleavage activity of the J4 and JFH-1 precursors was probed by western blotting using anti-NS3 polyclonal sera and anti-FLAG monoclonal antibody. Genotype 2a JFH-1 NS2-3 lacks the truncation product of genotype 1b J4 NS2-3 detected by anti-NS3 antibody. The black triangle indicates the NS3 cleavage product while the red triangle indicates the truncation product observed in J4 NS2-3. (B) Sequence alignment from Clustal W2 multiple alignment of genotype 1b strain J4 NS2-3 (GenBank accession no. AF054250) and genotype 2a strain JFH-1 NS2-3 (GenBank accession no. AB047639). The tryptophan fluorophores are highlighted in yellow and the cleavage site in cyan and active site residues in magenta. (C) The pH of the cleavage buffer was varied by 0.5 pH units within the range from pH 6.0 to pH 9.0 to assess the dependence of cleavage activity on pH for the two proteins. (D) The temperature dependence of cleavage activity between J4 and JFH-1 NS2-3 was compared over a 16-h period. Optimal activity at 24 °C for both proteins and inactivity of JFH-1 at 4 °C compared to J4 as observed. (E) The inhibition of auto-processing of NS2-3 by the chelation of metal ions was assessed by the addition of EDTA, EGTA and 1,10-phenanthroline (1,10-phen) to the cleavage reactions at 1 mM concentrations. (F) Wild-type NS2-3 precursor and the two active site mutants H956A and C997A were expressed and purified by nickel affinity chromatography and analysed by 15% SDS–PAGE followed by Coomassie staining. For auto-processing, purified wild-type NS2-3 precursor or the two mutants H956A and C997A were supplemented with 100 mM DTT, diluted 1:100 into cleavage buffer and incubated at 25 °C or 4 °C. For all data in panels C–F apart from the Coomassie stained gel in panel F, the products were separated by SDS–PAGE and probed by western blotting with anti-FLAG monoclonal antibody. Black triangles indicate the NS2-3 precursor and the NS3 protease domain cleavage product.
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Related In: Results  -  Collection

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fig2: Comparative analyses of recombinant WT NS2-3 precursor proteins derived from J4 or JFH-1 isolates. (A) The cleavage activity of the J4 and JFH-1 precursors was probed by western blotting using anti-NS3 polyclonal sera and anti-FLAG monoclonal antibody. Genotype 2a JFH-1 NS2-3 lacks the truncation product of genotype 1b J4 NS2-3 detected by anti-NS3 antibody. The black triangle indicates the NS3 cleavage product while the red triangle indicates the truncation product observed in J4 NS2-3. (B) Sequence alignment from Clustal W2 multiple alignment of genotype 1b strain J4 NS2-3 (GenBank accession no. AF054250) and genotype 2a strain JFH-1 NS2-3 (GenBank accession no. AB047639). The tryptophan fluorophores are highlighted in yellow and the cleavage site in cyan and active site residues in magenta. (C) The pH of the cleavage buffer was varied by 0.5 pH units within the range from pH 6.0 to pH 9.0 to assess the dependence of cleavage activity on pH for the two proteins. (D) The temperature dependence of cleavage activity between J4 and JFH-1 NS2-3 was compared over a 16-h period. Optimal activity at 24 °C for both proteins and inactivity of JFH-1 at 4 °C compared to J4 as observed. (E) The inhibition of auto-processing of NS2-3 by the chelation of metal ions was assessed by the addition of EDTA, EGTA and 1,10-phenanthroline (1,10-phen) to the cleavage reactions at 1 mM concentrations. (F) Wild-type NS2-3 precursor and the two active site mutants H956A and C997A were expressed and purified by nickel affinity chromatography and analysed by 15% SDS–PAGE followed by Coomassie staining. For auto-processing, purified wild-type NS2-3 precursor or the two mutants H956A and C997A were supplemented with 100 mM DTT, diluted 1:100 into cleavage buffer and incubated at 25 °C or 4 °C. For all data in panels C–F apart from the Coomassie stained gel in panel F, the products were separated by SDS–PAGE and probed by western blotting with anti-FLAG monoclonal antibody. Black triangles indicate the NS2-3 precursor and the NS3 protease domain cleavage product.
Mentions: Previous biochemical studies of the NS2-3 protease have been performed with protein of the genotype 1b J4 isolate. One complication of the analysis of the activity and folding of J4 NS2-3 is the presence of a 30-kDa C-terminal truncation product of the full-length protease (Fig. 2A) [13], which is detectable with NS3 antisera but not detectable with the FLAG antisera, as the truncation lacks the FLAG-tag located at the C-terminus of the full-length protein. This abundant truncation product is non-functional but would contribute significantly to the data collected in analyses of the global folding of the sample by tryptophan fluorescence. This truncation product, however, is not detectable in preparations of JFH-1 NS2-3, making the NS2-3 protease from genotype 2a more suitable for the subsequent assessment of refolding of the functional, full-length precursor state.

Bottom Line: The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage.We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage.Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

ABSTRACT
Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.

Show MeSH
Related in: MedlinePlus